Tembusu Virus

Overview and Taxonomy of Tembusu Virus

Historical Emergence and Initial Recognition

Tembusu virus (TMUV) represents a classic example of an emergent arthropod-borne virus that transitioned from relative obscurity to a pathogen of major agricultural significance. First isolated in 1955 from Culex tritaeniorhynchus mosquitoes in Malaysia, TMUV languished for decades as little more than a serological curiosity, overshadowed by more prominent flaviviruses such as dengue virus and Japanese encephalitis virus [32, 34]. Indeed, the virus was initially designated as part of the Tembusu serocomplex within the family Flaviviridae, but it garnered scant attention from the veterinary or medical research communities for over fifty years [32]. This quiescent period ended abruptly in April 2010, when an explosive outbreak of a previously unknown disease characterized by severe egg-drop syndrome and fatal neurological signs swept through duck farms in the major poultry-producing provinces of China, including Shandong, Jiangsu, and Zhejiang [13, 34]. The causative agent was rapidly identified as a flavivirus closely related to, but genetically distinct from, the original Malaysian TMUV isolate [34]. This event marked a watershed moment, transforming TMUV from an obscure mosquito virus into a globally significant emerging pathogen of poultry. The economic consequences were immediate and staggering; the 2010 epidemic caused a precipitous decline in egg production across affected flocks, with morbidity rates reaching 100% in some operations and mortality ranging from 5% to 30% depending on the age and immune status of the birds [13, 34]. Since that seminal outbreak, TMUV has spread relentlessly across Asia, establishing endemicity in China, Thailand, Vietnam, Malaysia, and Taiwan, and posing a persistent threat to the billions of dollars invested in the Asian waterfowl industry [15, 25, 31, 41].

Taxonomic Classification and Genomic Organization

TMUV is classified within the family Flaviviridae, genus Orthoflavivirus [1, 7, 9]. This taxonomic placement reflects its fundamental biological properties: a positive-sense, single-stranded RNA genome, a virion architecture characterized by a spherical, enveloped particle approximately 50 nm in diameter, and a replication cycle that is entirely cytoplasmic and intimately associated with host cell endoplasmic reticulum membranes [13, 29]. The viral genome is approximately 11 kb in length and encodes a single, long open reading frame (ORF) flanked by highly structured 5′ and 3′ untranslated regions (UTRs) that are critical for RNA replication, translation, and host-specific adaptation [13, 16, 40]. The polyprotein precursor is co- and post-translationally cleaved by a combination of host signal peptidases and the viral NS2B-NS3 serine protease to yield three structural proteins, capsid (C), pre-membrane (prM), and envelope (E), and seven non-structural (NS) proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [13, 24, 28]. The structural proteins constitute the physical virion and mediate host cell attachment and entry, with the E protein being the primary target of neutralizing antibodies and a major determinant of viral tropism and virulence [6, 9, 12, 42]. The non-structural proteins orchestrate the intracellular machinery of viral replication, RNA capping, and modulation of the host antiviral response [2, 3, 18, 24, 26, 28, 35, 36].

Phylogenetic Clustering and Genetic Diversity

Phylogenetic analyses employing full genome sequences and, most commonly, the envelope (E) gene have consistently resolved TMUV into three major genetic clusters, designated Cluster 1, Cluster 2, and Cluster 3, with Cluster 2 further subdivided into subclusters 2.1 and 2.2 [5, 8, 13, 25, 34]. More recent studies have proposed the existence of a fourth cluster (Cluster 4) to accommodate certain divergent strains, such as the Taiwanese mosquito isolate TP1906 and related goose-origin strains [27, 32]. The depth and breadth of this genetic diversity are striking and carry profound implications for viral pathogenesis, antigenic variation, and vaccine efficacy. Cluster 1 represents the earliest divergent lineage and includes the original Malaysian prototype strain (MM_1775) and several isolates from Thailand [5, 25, 34]. Cluster 1 viruses are generally considered less pathogenic in ducks compared to Cluster 2 strains, exhibiting slower replication kinetics, lower viral loads in target organs, and a reduced capacity for horizontal transmission [25, 33]. In experimental infections of Cherry Valley ducks, Cluster 1 DTMUV induced milder clinical signs, slower body weight loss, and lower morbidity and mortality rates compared with a reference Cluster 2.1 strain [25]. Furthermore, Cluster 1 displayed significant antigenic differences from Cluster 2.1, highlighting the potential for serological cross-reactivity challenges and the need for cluster-specific diagnostic and vaccine strategies [25].

Cluster 2 is the predominant and most intensively studied lineage, encompassing the vast majority of isolates responsible for the devastating outbreaks in China and Southeast Asia since 2010 [34, 41]. Within Cluster 2, subclusters 2.1 and 2.2 exhibit distinct epidemiological and pathogenic profiles. Cluster 2.1 is the dominant circulating lineage in Thailand and Vietnam and has also been identified in China [5, 15, 37]. This subcluster is highly pathogenic in ducks, causing acute egg-drop syndrome, severe neurological dysfunction, and significant mortality, particularly in young ducklings [5, 25]. Cluster 2.2, exemplified by strains such as Y and CHN-YC, has been associated with particularly high levels of neurovirulence in mammalian models, including mice [8, 9]. A detailed investigation into the molecular basis of virulence revealed that a single amino acid substitution at residue 487 in the E protein transmembrane domain (V487A) critically enhanced viral replication and neurovirulence in Kunming mice, a finding that underscores the capacity of minor genetic changes to profoundly alter viral pathogenicity [9]. The biological relevance of this residue was further underscored by its location within a domain implicated in flavivirus particle formation, suggesting that this substitution influences the efficiency of virion assembly and release [9].

Cluster 3 represents the most recently emerged lineage and has attracted intense scrutiny due to its expanding host range and unique transmission characteristics [11, 19, 21, 39]. First isolated from chickens and geese in China in the late 2010s and early 2020s, Cluster 3 viruses have been further subdivided into subgenotypes 3.1 and 3.2 [10, 22, 41]. The emergence of Cluster 3 strains is particularly concerning for several reasons. First, these viruses exhibit high infectivity and pathogenicity in chickens, a host that is relatively resistant to Cluster 2 strains [22, 39]. For example, the chicken-origin Cluster 3.2 strain TMUV-GX demonstrated high replication competence in multiple tissues of specific-pathogen-free (SPF) chicks, causing significant histopathological damage, whereas a duck-origin Cluster 2 strain (TMUV-JM) showed only low infectivity in the same avian species [22]. Second, Cluster 3 viruses have demonstrated the capacity for direct contact transmission in ducks, a property that facilitates rapid spread within dense poultry populations without the requirement for mosquito vectors [11]. Experimental co-housing studies with the Cluster 3 Shandong 2021 (SD) strain revealed that naïve ducks housed with infected ducks rapidly acquired the infection, with TMUV RNA detected in both throat and cloacal swabs [11]. In contrast, chickens infected with the same strain did not shed virus or transmit it to co-housed naïve chickens, indicating that while chickens are susceptible, they are likely a dead-end host for direct transmission [11]. This distinction is critical for understanding the epidemiology of Cluster 3 viruses: in ducks, direct contact transmission is a major driver of outbreaks, whereas in chickens, mosquito vectors such as Culex pipiens are essential for viral spread [11]. Third, Cluster 3 strains have been isolated from geese with high mortality rates, particularly in goslings, and have demonstrated the ability to cause severe neurological disease with mortality rates reaching 60–62.5% in some outbreaks [14, 19]. The GDE19-2024 strain, isolated from a goose farm in Guangdong Province, exhibited two distinct mutations at amino acid positions 149 and 157 of the E protein that set it apart from other Cluster 3 strains, suggesting ongoing adaptive evolution within this lineage [14].

Evolutionary Dynamics and Host Adaptation

A comprehensive analysis of TMUV evolution using codon usage patterns and phylogeographic reconstruction has provided valuable insights into the forces shaping the virus's emergence and diversification. The time to the most recent common ancestor (tMRCA) of all contemporary TMUV strains has been estimated to be approximately 1924, predating its first isolation by three decades and suggesting a long period of cryptic circulation in sylvatic cycles before its emergence in domestic poultry [34]. The effective population size of TMUV underwent a dramatic expansion from 2010 to 2013, coinciding precisely with the first major duck outbreaks in China and the subsequent diversification of the major clusters [34]. Phylogeographic analyses strongly suggest that Malaysia is the most likely geographic origin of the virus, while Shandong Province in China was the earliest site of introduction and subsequent epidemic spread within mainland China [34]. The pattern of emergence points to long-distance dissemination, potentially via migratory birds along the East Asian-Australasian Flyway, followed by spillover into local mosquito populations and domestic poultry [20]. Indeed, surveillance on Chongming Island, a key stopover site for migratory birds, detected Cluster 3 TMUV strains that were closely related to viruses from Southeast Asia but distant from the original 2010 outbreak strains, providing direct evidence for the role of migratory birds in the transboundary dispersal of the virus [20].

Codon usage analysis has revealed that natural selection is the predominant force shaping the TMUV genome [17]. The virus exhibits a codon adaptation index (CAI) that is markedly higher for its primary avian hosts (ducks, geese, and chickens) than for potential mammalian hosts (humans) or mosquito vectors [17]. This species-specific adaptation is a critical determinant of host range, infectivity, and clinical outcome. Further, the TMUV genome displays significant suppression of CpG and UpA dinucleotides, a pattern commonly observed in vertebrate RNA viruses and attributed to selective pressure to evade recognition by host innate immune sensors, particularly the ZAP (zinc finger antiviral protein) system [17]. The progressive alignment of TMUV with host codon usage patterns and the continuous reduction in CpG dinucleotides over time suggest that the virus is becoming increasingly well-adapted to its avian hosts, a phenomenon that has implications for both viral fitness and pathogenicity [17].

Expanding Host Range and Zoonotic Potential

The taxonomy and evolutionary history of TMUV cannot be understood without appreciating its extraordinary capacity for host switching. Originally isolated from mosquitoes, the virus has demonstrated the ability to infect an increasingly diverse array of vertebrate species, including ducks, geese, chickens, pigeons, mice, and, most alarmingly, marine mammals [4, 8, 13, 19, 23, 30, 33]. The host range expansion is not merely a laboratory phenomenon; it has been documented in natural settings. In July 2023, a fatal outbreak of TMUV infection occurred in three zoo-housed bottlenose dolphins (Tursiops truncatus) in Thailand, marking the first documented natural infection of TMUV in a mammal outside of laboratory conditions [4]. The dolphins presented with severe neurological symptoms, and postmortem analysis confirmed high viral loads in brain and lung tissues, with viral antigen specifically localized to neurons and astroglial cells [4]. Phylogenetic analysis placed the dolphin TMUV strains within Cluster 3, closely related to strains found in mosquitoes in China, and retrospective analysis of dolphin samples from 2019 confirmed persistent circulation of the virus in the captive population [4]. This event has profound implications for our understanding of TMUV's zoonotic potential and underscores the need for a One Health approach to surveillance and risk assessment [4].

Concerns about human infection are not hypothetical. Serological surveys of duck farm workers in China and Thailand have detected the presence of neutralizing antibodies against TMUV at rates significantly higher than those in the general population without duck contact [13, 38]. A 2021 study in central Thailand demonstrated that sera from an at-risk population (duck farm workers and residents of farming areas) had higher anti-DTMUV neutralizing antibody titers compared to a not-at-risk population living distant from farms [38]. Crucially, these sera showed no or low cross-reactivity with other endemic flaviviruses, suggesting that the neutralizing activity was specific to TMUV [38]. While no clinical disease has yet been causally linked to TMUV in humans, the detection of both viral RNA and specific antibodies in individuals with occupational exposure provides strong evidence for subclinical or mild infection and highlights the potential for this virus to spill over into human populations [13, 38]. The World Health Organization (WHO) and the World Organisation for Animal Health (WOAH) have increasingly emphasized the need for integrated surveillance of emerging flaviviruses at the human-animal-ecosystem interface, and TMUV represents a prime candidate for such surveillance given its demonstrated capacity for interspecies transmission and its genetic plasticity.

Molecular Pathogenesis: NS1 Protein Degradation and Innate Immune Evasion

The capacity of Tembusu virus (TMUV) to establish a productive infection in its avian hosts, and increasingly, in mammalian species, is fundamentally dependent on its ability to subvert the host’s intrinsic antiviral defenses. Central to this host-pathogen arms race is the viral nonstructural protein 1 (NS1), a multifunctional glycoprotein that plays critical roles in viral RNA replication, immune evasion, and pathogenesis. While NS1 is well-characterized in other flaviviruses for its role in modulating complement activation and endothelial barrier dysfunction, the molecular mechanisms governing NS1 stability and its interplay with the duck innate immune system have only recently been elucidated. The discovery that host restriction factors can directly target NS1 for proteasomal degradation, coupled with the virus’s own countermeasures to dismantle interferon (IFN) signaling, reveals a sophisticated layer of regulation at the heart of TMUV pathogenesis. This section provides an exhaustive analysis of the molecular mechanisms by which TMUV NS1 is targeted for degradation and how the virus, in turn, orchestrates a multi-pronged evasion of the type I interferon response, drawing on a wealth of recent structural and functional studies.

The TRIM14-NS1 Axis: A Paradigm of Host Restriction via K27/K29-Linked Ubiquitination

Among the most significant advances in understanding TMUV innate immune evasion is the characterization of duck tripartite motif-containing protein 14 (duTRIM14) as a potent, dual-action host restriction factor. TRIM proteins are a large family of E3 ubiquitin ligases that regulate diverse cellular processes, including antiviral immunity. Zhou et al. (2025) demonstrated that duTRIM14 expression is critical for controlling TMUV replication; its overexpression significantly suppresses viral titers, while its deficiency markedly enhances viral proliferation [2]. The mechanistic basis for this restriction lies in a direct physical interaction between duTRIM14 and the viral NS1 protein. This interaction facilitates the conjugation of K27- and K29-linked polyubiquitin chains onto NS1, a non-canonical ubiquitin linkage type that is increasingly recognized for its role in targeting proteins for proteasomal degradation [2]. Critically, the lysine residue at position 141 (Lys141) on NS1 was identified as the primary acceptor site for this ubiquitination. Mutagenesis of this single residue (K141R) rendered NS1 resistant to duTRIM14-mediated degradation, resulting in a virus that exhibited significantly enhanced replication both in vitro and in vivo [2]. This finding underscores the exquisite specificity of the host-virus interaction and identifies NS1 Lys141 as a critical vulnerability in the TMUV life cycle.

The functional significance of this degradation extends beyond merely reducing the pool of NS1 available for replication complex formation. By eliminating NS1, duTRIM14 simultaneously removes a key viral antagonist of the host immune response. NS1 has been shown in other flavivirus systems to inhibit complement activation and modulate Toll-like receptor (TLR) signaling. Therefore, the TRIM14-mediated degradation of NS1 serves a dual purpose: it directly impairs viral RNA replication, which is dependent on NS1, and it relieves NS1-mediated suppression of the host’s antiviral state. Remarkably, duTRIM14 does not stop at degrading NS1. Zhou et al. (2025) further demonstrated that duTRIM14 interacts with duck TANK-binding kinase 1 (duTBK1), a master kinase in the RIG-I-like receptor (RLR) signaling pathway that leads to IFN-β production. This interaction promotes K63-linked polyubiquitination of duTBK1 at residues Lys30 and Lys401, a modification that is essential for TBK1 activation and its subsequent phosphorylation of interferon regulatory factor 3 (IRF3) and IRF7 [2]. The result is a potent augmentation of IFN-β production. Thus, duTRIM14 acts as a molecular fulcrum, simultaneously tipping the balance away from viral replication by degrading NS1 and toward host defense by amplifying TBK1-dependent interferon signaling. This dual-action mechanism represents a highly efficient host strategy to combat TMUV infection.

Counterpoint: The Viral Offensive, NS2B-Mediated Degradation of MAVS and the JOSD1-SOCS1-IRF7 Axis

While the host deploys TRIM14 to degrade viral proteins, TMUV has evolved its own sophisticated machinery to dismantle the host’s interferon induction pathway. The viral nonstructural protein 2B (NS2B), a component of the NS2B-NS3 protease complex, has been identified as a potent antagonist of type I interferon production. Zhou et al. (2022) revealed that TMUV NS2B directly targets the mitochondrial antiviral-signaling protein (MAVS), a central adaptor molecule that links RIG-I/MDA5 activation to downstream TBK1 and IKKε signaling [35]. NS2B interacts specifically with duck MAVS (duMAVS) and recruits the cellular E3 ubiquitin ligase duck membrane-associated RING-CH-type finger 5 (duMARCH5). This recruitment leads to the conjugation of K48-linked polyubiquitin chains onto duMAVS at residues K321, K354, K398, and K411, marking it for proteasomal degradation [35]. By eliminating MAVS, NS2B effectively severs the connection between viral RNA sensing and the activation of IRF3/7, thereby blocking the transcription of IFN-β. This is a highly effective strategy, as it prevents the initial wave of interferon production that would otherwise establish an antiviral state in neighboring cells.

Beyond the MAVS node, TMUV employs a second, parallel strategy to suppress interferon signaling by targeting the transcription factors themselves. The suppressor of cytokine signaling 1 (SOCS1) is a well-known negative regulator of cytokine signaling, and its expression is often hijacked by viruses to dampen immune responses. Huang et al. (2022) demonstrated that TMUV infection upregulates SOCS1 expression through the activation of Toll-like receptor 3 (TLR3) signaling [36]. This upregulation is further stabilized by the host deubiquitinase JOSD1, which binds to the SH2 domain of SOCS1 and removes its K48-linked ubiquitin chains, preventing its own proteasomal degradation [36]. The stabilized SOCS1 then acts as an E3 ubiquitin ligase, binding directly to interferon regulatory factor 7 (IRF7) and catalyzing its K48-linked ubiquitination and subsequent degradation [36]. Since IRF7 is a master regulator of type I interferon production, particularly in the amplification phase of the response, its degradation effectively cripples the cell’s ability to mount a robust IFN response. This JOSD1-SOCS1-IRF7 negative-feedback loop represents a sophisticated viral strategy to not only block initial IFN induction (via NS2B-MAVS) but also to dismantle the amplification loop that would otherwise sustain the antiviral state.

The NS5-TRAF3 Interface and the MARCH6-NS5-TOLLIP Autophagic Axis

The viral RNA-dependent RNA polymerase, NS5, is another prime target for host restriction and a key player in immune evasion. Cai et al. (2023) demonstrated that duck TRAF3 (duTRAF3), a critical adaptor protein in the RIG-I signaling pathway, acts as a potent antiviral factor against TMUV. Overexpression of duTRAF3 inhibits DTMUV replication in a dose-dependent manner and activates the transcription of IFN-α and downstream interferon-stimulated genes (ISGs) [26]. However, TMUV NS5 directly counteracts this by interacting with duTRAF3 and inhibiting its expression, thereby neutralizing the antiviral signal [26]. This interaction highlights a direct viral countermeasure against a central innate immune adaptor.

In a striking parallel to the TRIM14-NS1 degradation mechanism, the host has evolved an alternative strategy to target NS5 for destruction, but through a completely different pathway. Zhou et al. (2025) discovered that the E3 ligase membrane-associated RING finger 6 (MARCH6) is significantly upregulated during TMUV infection and acts as a potent restriction factor [3]. Unlike TRIM14, MARCH6 suppresses TMUV replication through an E3 ligase activity-independent mechanism. Instead, MARCH6 directly interacts with NS5 and recruits the autophagic cargo receptor TOLLIP. This MARCH6-NS5-TOLLIP interaction directs NS5 to nascent phagophores for degradation via selective autophagy, bypassing the need for conventional ubiquitin signaling [3]. This is a groundbreaking finding, as it reveals a novel host defense strategy where a viral polymerase is targeted for autophagic degradation by a cargo receptor that does not require ubiquitin as a recognition signal. The MARCH6-NS5-TOLLIP axis represents a critical, previously uncharacterized arm of the host’s antiviral arsenal, demonstrating that selective autophagy is a fundamental mechanism for controlling TMUV infection.

Broader Implications for Innate Immune Subversion and Viral Pathogenesis

The interplay between NS1 degradation and innate immune evasion is not an isolated event but is deeply integrated into the broader pathogenesis of TMUV. The virus’s ability to manipulate cellular stress responses, such as autophagy and apoptosis, further complicates the host-pathogen interaction. TMUV infection induces both complete and incomplete autophagy in different cell types, a process that the virus hijacks to promote its own replication. Tan et al. (2023) showed that the nonstructural proteins NS2B and NS4A induce complete autophagy by interacting with the cargo receptor p62/SQSTM1, which facilitates viral replication [24]. Conversely, Wang et al. (2023) demonstrated that in neuronal cells, DTMUV induces incomplete autophagy via the ERK/mTOR and AMPK/mTOR signaling pathways, which also promotes viral replication and contributes to neuropathogenesis [43]. The induction of apoptosis is another critical facet. Pan et al. (2023) revealed that the NS3 protein is the main inducer of apoptosis, activating the PERK/PKR pathway and interacting with voltage-dependent anion channel 2 (VDAC2) to trigger the mitochondrial apoptotic pathway [28]. This virus-induced apoptosis may facilitate viral dissemination while simultaneously contributing to tissue pathology, such as the testicular atrophy and ovarian damage observed in infected ducks [44, 45].

Furthermore, the virus’s ability to evade innate immunity is directly linked to its expanding host range and increasing virulence. The emergence of cluster 3 strains, which exhibit enhanced neurovirulence in mice and can be transmitted via aerosols, underscores the adaptive potential of TMUV [8, 9, 11, 19]. A single amino acid substitution in the E protein (V487A) was shown to significantly enhance viral assembly and neurovirulence in mice, highlighting how minor genetic changes can dramatically alter pathogenesis [9]. The virus’s capacity to replicate in mosquito vectors, and the role of mosquito salivary proteins like the 34-kDa protein in suppressing the vector’s innate immune response, further illustrates the multifaceted nature of TMUV’s immune evasion strategies [5, 46]. The detection of TMUV antibodies in duck farm workers and the first documented natural fatal infection in bottlenose dolphins in Thailand serve as stark reminders of the zoonotic potential of this virus, emphasizing the critical need to understand the molecular underpinnings of its immune evasion [4, 38]. The World Organisation for Animal Health (WOAH) recognizes TMUV as a significant transboundary animal disease, and the Food and Agriculture Organization (FAO) has highlighted its economic impact on the poultry sector in Asia, underscoring the global relevance of this research.

Epidemiology and Transmission Dynamics of Tembusu Virus

Tembusu virus (TMUV), an emerging avian orthoflavivirus within the family Flaviviridae, has transitioned from a relatively obscure mosquito-borne agent to a pathogen of significant economic and ecological consequence across Asia. Since its initial isolation from Culex tritaeniorhynchus mosquitoes in Malaysia in 1955 [34], TMUV remained largely uncharacterized for decades. However, the landscape of TMUV epidemiology was irrevocably altered in April 2010, when a massive outbreak of acute egg-drop syndrome and fatal encephalitis erupted in duck farms along the southeastern coast of China, rapidly spreading to most major duck-producing provinces [13, 34]. This event marked the emergence of a virus with dramatically altered transmission dynamics, including the acquisition of direct contact and aerosol transmission routes, a phenomenon rarely observed among mosquito-borne flaviviruses [11]. The subsequent rapid dissemination of TMUV across Asia, its expanding host range encompassing multiple avian species and now mammals, and the detection of neutralizing antibodies in humans with occupational exposure, underscore a pressing need for comprehensive epidemiological surveillance and a One Health approach to mitigate its potential threat to both animal and public health [4, 13, 32, 38].

Geographic Distribution and Phylogenetic Clusters

The global epidemiology of TMUV is characterized by a complex phylogeographic structure, with the virus now classified into three major genetic clusters (Clusters 1, 2, and 3) based on the envelope (E) gene sequence, with Cluster 2 further subdivided into subclusters 2.1 and 2.2 [5, 34, 39]. Phylogeographic analyses have pinpointed Malaysia as the most likely ancestral origin of TMUV, with the time to the most recent common ancestor estimated to be around 1924, predating its first isolation by several decades [34]. From this epicenter, the virus has radiated outwards, with Shandong Province in China identified as the earliest point of introduction and subsequent diversification within the country [34].

Cluster 2 has emerged as the dominant and most widely distributed lineage, responsible for the major epizootics in ducks across China, Thailand, and Vietnam since 2010 [17, 34]. Within this cluster, subcluster 2.2 is the most prevalent globally, while subcluster 2.1 is the predominant circulating genotype in Thailand and has been consistently isolated in Vietnam [15, 25, 37]. The epidemiological success of Cluster 2 is partly attributed to its superior adaptation to the codon usage patterns of domestic ducks, the primary amplifying host, suggesting that natural selection has played a pivotal role in shaping its evolutionary trajectory [17]. In contrast, Cluster 1 strains, which are generally less pathogenic in ducks, have been reported in Thailand and China, though their circulation appears more restricted [25, 34].

The most recent and concerning epidemiological development is the emergence and expansion of Cluster 3 TMUV. Initially identified from mosquito isolates in Yunnan Province, China, in 2012 [52], Cluster 3 strains have since been increasingly isolated from chickens, geese, and ducks, causing significant outbreaks [10, 11, 14, 19, 21, 39]. A landmark event was the isolation of Cluster 3.2 strains from laying hens in Guangdong, China, in 2020, which caused severe egg-drop syndrome and demonstrated significant antigenic variation compared to Cluster 2 strains [22, 39]. This cluster has now been detected across a wide geographic range, including China, Thailand, and Taiwan, and has been linked to long-distance dispersal events [20, 49, 53]. The detection of Cluster 3 TMUV in migratory bird fecal samples on Chongming Island, China, provides compelling evidence for the role of avian migration in the intercontinental spread of the virus, potentially introducing Southeast Asian genotypes into new regions [20]. The continuous evolution of TMUV is further evidenced by the identification of novel subgenotypes, such as subgenotype 2.1.1 in goslings in China and subgenotype 3.2 in geese, highlighting the ongoing genetic diversification and adaptation of the virus to new hosts and ecological niches [10, 41].

Host Range and Species Susceptibility

While domestic ducks (Anas platyrhynchos domesticus) remain the primary amplifying host and the species most severely affected by TMUV, the virus has demonstrated a remarkable and expanding host range that now includes multiple avian species and, alarmingly, mammals [13, 32].

Avian Hosts: The susceptibility and clinical outcome of TMUV infection vary dramatically among avian species. In ducks, the virus causes acute egg-drop syndrome in laying adults and severe neurological dysfunction with high mortality in ducklings, with the severity of disease being strongly age-dependent [13, 48]. Younger ducks (e.g., 4-week-old) exhibit more severe disease and higher mortality than older ducks (e.g., 27-week-old), a phenomenon linked to age-related differences in immune responses, including a more robust neutralizing antibody and cytotoxic T lymphocyte response in older birds [48, 57]. Pathogenicity is also genotype-dependent; Cluster 2.1 strains are generally more pathogenic in ducks than Cluster 1 strains, causing higher morbidity, mortality, and more severe pathological lesions [25]. Geese have emerged as a highly susceptible and increasingly important host. Cluster 3 strains, such as TMUV HQ-22 and GDE19-2024, have been shown to cause mortality rates as high as 60-62.5% in goslings, with severe neurological signs and hemorrhagic lesions, suggesting a particular adaptation of this cluster to geese [14, 19]. Novel subgenotype 2.1.1 strains have also been associated with high mortality (~50-60%) in goslings in China [41]. In contrast, chickens were initially considered less susceptible, with Cluster 2 strains showing low infectivity and pathogenicity in chicks [22]. However, the emergence of Cluster 3.2 strains has changed this paradigm. Chicken-origin Cluster 3.2 TMUV exhibits high replication competence in multiple tissues of chicks, causing histopathological damage and egg-drop syndrome in laying hens, indicating that this cluster has a biological basis for widespread infection in chickens [22, 39]. Furthermore, experimental evidence has confirmed direct contact transmission of a goose-origin TMUV strain among day-old chicks, underscoring the potential for rapid spread within chicken populations [23].

Mammalian Hosts and Zoonotic Potential: The capacity of TMUV to infect mammals has been a critical focus of recent research. Experimental infections have demonstrated that mice (BALB/c and Kunming) are susceptible to TMUV, particularly via the intracerebral (i.c.) route, where the virus causes lethal neurological disease [8, 9, 30]. Importantly, Cluster 3 strains, such as TMUV HQ-22, have been shown to cause severe neurological symptoms and mortality in mice via both i.c. and intranasal routes, raising concerns about airborne transmission to mammals [19]. The neurovirulence of TMUV in mice is linked to specific genetic determinants, including a single amino acid substitution (V487A) in the E protein transmembrane domain, which enhances viral assembly and replication in the brain [9]. The mosquito-derived MM 1775 strain has also been shown to exhibit higher pathogenicity in mice compared to duck-derived strains, suggesting that viral origin influences mammalian virulence [33].

The most significant evidence for zoonotic risk comes from serological surveys of human populations. A seminal study in Thailand detected significantly higher neutralizing antibody titers against a local DTMUV isolate in duck farm workers and residents of farming areas (the at-risk population) compared to individuals with no duck contact [38]. Critically, some individuals in the not-at-risk group also displayed high neutralizing titers, suggesting potential alternative routes of exposure or vector-borne transmission to humans [38]. These findings corroborate earlier reports from China, where viral RNA and antibodies were detected in duck industry workers [13, 28]. The first documented natural fatal infection of TMUV in a mammal was reported in 2023, when three bottlenose dolphins in a Thai zoo succumbed to the virus, exhibiting severe neurological symptoms and high viral loads in brain and lung tissues [4]. Phylogenetic analysis placed the dolphin strains within Cluster 3, closely related to mosquito isolates from China, highlighting the virus's ability to jump across substantial phylogenetic barriers [4]. This event, combined with the human serological data, mandates that TMUV be recognized as a potential zoonotic pathogen requiring active surveillance under a One Health framework, as advocated by the World Organisation for Animal Health (WOAH) and the World Health Organization (WHO).

Transmission Dynamics: From Vector-Borne to Direct Contact

The transmission biology of TMUV is a critical factor in its epidemic success and represents a significant departure from classical flavivirus transmission. While TMUV is fundamentally a mosquito-borne virus, its ability to transmit via direct contact and aerosol routes has been a key driver of its rapid spread in intensive poultry farming systems [11, 58].

Vector-Borne Transmission: The primary vectors for TMUV are mosquitoes of the Culex genus, particularly Culex tritaeniorhynchus and Culex quinquefasciatus [5, 11, 16]. Aedes albopictus and Aedes aegypti have also been demonstrated to be competent vectors, which is significant given their preference for feeding on mammals, including humans [30, 46]. The vector competence of different mosquito species and the efficiency of transmission are influenced by both viral and vector factors. For instance, the prototypical mosquito-derived TMUV strain MM_1775 exhibits significantly higher infectivity and transmission rates in Cx. quinquefasciatus compared to the duck-derived CQW1 strain, with the viral E protein and 3′ untranslated region (UTR) identified as key determinants of this difference [16]. Furthermore, different TMUV genotypes show distinct interactions with vectors. In Cx. tritaeniorhynchus, Cluster 1 DTMUV undergoes higher replication in salivary glands and saliva compared to Cluster 2.1, potentially leading to more efficient transmission [5]. The virus can also be maintained within mosquito populations through vertical (transovarial) and venereal transmission, ensuring its persistence even during inter-epidemic periods [16]. The mosquito's innate immune system plays a crucial role in modulating infection; for example, a 34-kDa salivary protein in Ae. albopictus enhances DTMUV infectivity by suppressing the antiviral immune response in the salivary glands [46].

Direct Contact and Aerosol Transmission: The 2010 outbreak in China revealed a paradigm shift in TMUV transmission. Unlike most flaviviruses, the epidemic strains had acquired the ability to spread directly between birds without a mosquito vector [11, 58]. Experimental studies have confirmed that Cluster 2 and Cluster 3 TMUV strains can be transmitted from infected to naïve ducks through direct contact and, critically, via the aerosol route [11, 23]. This is facilitated by the shedding of high titers of virus through the respiratory tract (throat swabs) and digestive tract (cloacal swabs) in ducks [11]. The ability to transmit directly is a major factor explaining the explosive nature of outbreaks in densely populated duck farms. Interestingly, this direct transmission capability appears to be host-species specific. While ducks shed virus and transmit it directly, chickens infected with the same Cluster 3 strain do not shed detectable virus and cannot transmit the virus to co-housed naïve chickens, requiring mosquitoes for transmission [11]. This suggests that the virus's transmission route is contingent upon the host species, with ducks serving as the primary amplifier for direct spread. The molecular basis for this difference in shedding is an active area of research, but it is linked to the virus's ability to replicate in the gastrointestinal tract. The gut microbiome, particularly Gram-negative bacteria and their lipopolysaccharide (LPS), has been shown to enhance TMUV proliferation in the intestine by promoting viral attachment via Toll-like receptor 4 (TLR4), providing a mechanistic link between the microbiota and fecal-oral transmission [58].

Epidemiological Drivers and Risk Factors

Several interconnected factors drive the epidemiology of TMUV, creating a complex web of risk that facilitates its emergence and spread.

1. Agricultural Intensification: The rapid expansion and intensification of duck farming in Asia, particularly in China and Southeast Asia, has created ideal conditions for TMUV transmission. High-density flocks, continuous production cycles, and the commingling of different age groups on farms provide a constant supply of susceptible hosts and amplify viral shedding [13, 34].
2. Host Age and Immunity: Age is a critical determinant of disease severity. Young ducklings (2-4 weeks old) are highly susceptible to severe neurological disease and mortality, while adult laying ducks experience a dramatic drop in egg production but lower mortality [15, 48]. This age-dependent susceptibility is linked to the maturation of the adaptive immune system, with older ducks mounting more effective neutralizing antibody and cytotoxic T cell responses [48, 57].
3. Viral Genotype and Evolution: The continuous emergence of new genetic clusters and subclusters with distinct pathogenic and antigenic profiles is a major driver of epidemiological change. The dominance of Cluster 2, the emergence of the highly pathogenic Cluster 3 in chickens and geese, and the identification of novel subgenotypes all point to rapid viral evolution [17, 22, 41]. The presence of positive selection on codons in the NS3 and NS5 genes suggests ongoing adaptation to enhance replication fitness [34]. Antigenic variation between clusters, as demonstrated by cross-neutralization tests, poses a challenge for vaccine efficacy, as vaccines developed against one cluster may not provide complete protection against another [25, 39].
4. Vector Ecology and Climate: The distribution and abundance of mosquito vectors, particularly Culex spp., are heavily influenced by climatic factors such as temperature, rainfall, and humidity. The persistence of TMUV in mosquitoes through vertical transmission allows it to overwinter and re-emerge in the spring [16, 52]. The detection of TMUV in mosquitoes in northern Thailand and Yunnan Province, China, highlights the wide geographic range of enzootic circulation [52, 53].
5. Wildlife and Migratory Birds: The role of migratory birds in the long-distance dispersal of TMUV is increasingly recognized. The detection of TMUV RNA in fecal samples from migratory birds on Chongming Island, a critical stopover site on the East Asian-Australasian Flyway, provides direct evidence for this mechanism [20]. This allows the virus to be introduced into new geographic regions, potentially seeding outbreaks in naïve poultry populations.
6. Biosecurity Gaps: The ability of TMUV to transmit via direct contact and aerosol routes means that standard vector control measures are insufficient to prevent farm-to-farm spread. Inadequate biosecurity, including the movement of infected birds, contaminated equipment, and personnel, facilitates the rapid dissemination of the virus [11].

Surveillance and Diagnostic Challenges

Effective epidemiological control of TMUV relies on robust surveillance and rapid, accurate diagnosis. The development of highly sensitive and specific molecular tools has been a priority. Multiplex real-time RT-qPCR assays have been developed to simultaneously detect DTMUV alongside other common duck pathogens like novel duck reovirus (NDRV) and duck hepatitis A virus (DHAV), enabling differential diagnosis in clinical settings [1]. Digital PCR (dPCR) offers even higher sensitivity, with detection limits as low as 1.3 copies/μL, outperforming qPCR for detecting low-level infections [51]. For field-deployable diagnostics, isothermal amplification methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with CRISPR/Cas12a or Cas13a systems provide rapid, visual, and highly specific detection without the need for sophisticated laboratory equipment [47, 54-56]. These point-of-care tools are invaluable for early outbreak detection in resource-limited settings.

Serological surveillance is complicated by the high degree of antibody cross-reactivity among flaviviruses, particularly with Japanese encephalitis virus (JEV) [7]. To overcome this, a highly specific enzyme-linked immunosorbent assay (ELISA) using subviral particles (SPs) expressing the prM and E proteins of DTMUV has been developed, demonstrating 100% sensitivity and specificity compared to the serum neutralization test and no cross-reactivity with JEV antibodies [7]. This assay is a powerful tool for large-scale serosurveys to monitor virus circulation and vaccine efficacy. Despite these advances, significant surveillance gaps remain. Many countries in Southeast Asia lack systematic surveillance programs, and the true geographic distribution and prevalence of TMUV, particularly in wild bird populations and potential mammalian hosts, are likely underestimated [50, 53]. The absence of TMUV in South Korea, despite its location on a major migratory bird flyway, highlights the potential for successful prevention through stringent biosecurity, but also underscores the constant risk of introduction [50]. The detection of TMUV in zoo dolphins in Thailand [4] and neutralizing antibodies in humans [38] serves as a stark reminder that surveillance must extend beyond poultry to include wildlife and human populations at the animal-human interface, aligning with the principles of a One Health approach championed by the WHO, WOAH, and FAO.

Clinical Manifestations and Pathology in Ducks and Geese

Tembusu virus (TMUV) infection in domestic waterfowl presents a complex and highly variable clinical picture, heavily influenced by viral genotype, host species, age, and immune status. The disease is characterized by two primary syndromic presentations: acute egg-drop syndrome in adult laying birds and severe neurological dysfunction in young ducklings and goslings [13, 32]. The economic impact is profound, with morbidity rates in affected flocks often reaching 90-100% and mortality rates varying dramatically from 5-30% in typical duck infections to over 60% in highly pathogenic goose outbreaks [13, 41]. The virus has been classified into three distinct genetic clusters (1, 2, and 3), with subclusters within cluster 2 (2.1 and 2.2) and cluster 3 (3.1 and 3.2) exhibiting marked differences in pathogenicity and tissue tropism [5, 25, 34]. Understanding these genotype-phenotype correlations is critical for accurate diagnosis, surveillance, and vaccine strain selection.

Clinical Manifestations in Ducks

Neurological Signs in Ducklings: In young ducklings, particularly those aged 2-4 weeks, TMUV infection manifests as an acute, rapidly progressive neurological syndrome [15]. The incubation period is typically 3-5 days post-infection. Initial signs include lethargy, anorexia, and reluctance to move, rapidly progressing to overt neurological deficits such as ataxia, incoordination, tremors of the head and neck, and opisthotonos [13, 25]. Affected ducklings often lie on their sides with paddling movements of the legs. Morbidity is high, and mortality can reach 30-50% in severe outbreaks, with death typically occurring within 48-72 hours of neurological onset [25]. Importantly, cluster 2.1 strains consistently induce more severe neurological disease and higher mortality in ducklings compared to cluster 1 strains, which tend to cause milder, transient signs with lower mortality rates [25]. This differential pathogenicity is correlated with higher and more sustained viremia, earlier viral dissemination to the central nervous system (CNS), and more pronounced histopathological lesions in cluster 2.1-infected birds [25].

Egg-Drop Syndrome in Adult Laying Ducks: In adult laying ducks, the hallmark clinical sign is a dramatic and acute drop in egg production, often plummeting from peak production (90-95%) to 10-20% within 3-5 days [13, 32]. This is frequently accompanied by the production of abnormal eggs, including soft-shelled, thin-shelled, misshapen, and pale eggs [13, 39]. Affected hens may exhibit mild to moderate anorexia, depression, and ruffled feathers, but neurological signs are less common and typically milder than in ducklings, often limited to transient ataxia or head tremors [48]. The severity of egg drop is directly correlated with viral replication in the reproductive tract, particularly the ovary and oviduct, leading to follicular degeneration, hemorrhage, and atresia [13, 44]. Recovery of egg production is slow and often incomplete, with many flocks failing to return to pre-infection production levels, resulting in long-term economic losses [13].

Age-Related Differences in Disease Severity: A critical aspect of TMUV pathogenesis in ducks is the profound age-dependent susceptibility. Young ducks (4-week-old) exhibit severe, often fatal neurological disease with high viral loads in the brain and spleen, while adult laying ducks (27-week-old) develop a milder, primarily reproductive syndrome with lower mortality [48]. This age-related resistance is underpinned by distinct immunological mechanisms. Adult ducks mount a more robust and coordinated adaptive immune response, characterized by a strong cytotoxic T lymphocyte (CTL) response that significantly correlates with the reduction of viremia and viral loads in target organs [48]. Furthermore, adult ducks produce high levels of neutralizing antibodies that effectively clear the virus from the blood and tissues [48, 57]. In contrast, young ducks exhibit a dysregulated innate immune response, with a pronounced loss of non-T and B lymphocytes (myeloid cells) and impaired phagocytic activity during the critical early phase of infection (3-5 days post-infection), which correlates with uncontrolled viral replication and severe disease [48, 57]. This age-related immunopathology highlights the critical role of a balanced and timely immune response in controlling TMUV infection.

Clinical Manifestations in Geese

TMUV infection in geese has emerged as a significant and increasingly recognized problem, particularly with the emergence and spread of cluster 3 strains [14, 19, 41]. The clinical picture in geese can be distinctly more severe than in ducks, with higher mortality rates and unique pathological features.

High Mortality in Goslings: Goslings, especially those under 2 weeks of age, are highly susceptible to TMUV, with mortality rates often exceeding 50% and reaching up to 62.5% in some outbreaks [14, 41]. The clinical course is rapid and fulminant. Infected goslings develop severe neurological signs, including ataxia, tremors, paralysis, and opisthotonos, often within 2-4 days of infection [14, 19, 59]. Anorexia, depression, and reluctance to move are common prodromal signs. The disease progresses quickly, with death occurring within 24-48 hours of neurological onset. This high mortality is associated with extensive viral replication in multiple organs, particularly the brain, liver, and spleen, leading to severe histopathological damage [14, 41, 59]. Cluster 3 strains, such as GDE19-2024 and HQ-22, appear to be particularly adapted to geese, exhibiting higher pathogenicity in this species compared to ducks [14, 19].

Reproductive Failure in Adult Geese: In adult laying geese, TMUV infection causes a severe and acute egg-drop syndrome, mirroring that seen in ducks but often with more profound and prolonged effects [10, 21]. Affected flocks experience a precipitous decline in egg production, often accompanied by the production of abnormal eggs. Ovarian pathology is severe, with extensive follicular hemorrhage, degeneration, and atresia [21]. The virus has been isolated from geese with ovaritis, confirming its direct role in reproductive tract pathology [21]. In some outbreaks, particularly those caused by novel cluster 3 strains, mortality in adult geese can also be elevated, a feature less commonly observed in adult ducks [10, 21].

Gross Pathology

Nervous System: The most consistent gross lesion in both ducks and geese is splenomegaly, often with a mottled or marbled appearance [13, 25]. The brain may appear grossly normal or show mild congestion and edema, particularly in the cerebrum and cerebellum [13, 45]. In severe cases, especially in goslings, petechial hemorrhages may be observed on the surface of the brain [14].

Reproductive Tract: In laying females, the ovaries are the primary target organ. Gross lesions include severe follicular hemorrhage, with follicles appearing congested, hemorrhagic, and atretic [13, 39, 44]. The oviduct may be edematous and congested. In males, testicular atrophy and degeneration have been reported, with histopathological evidence of vacuolation and apoptosis of spermatogenic cells [44].

Other Visceral Organs: Hepatomegaly and splenomegaly are common findings [13, 23]. The liver may appear pale, friable, or mottled. The spleen is often enlarged and congested. In some cases, particularly with highly pathogenic goose strains, hemorrhagic lesions may be observed in the lungs, kidneys, and intestinal mucosa [14]. Myocardial pallor or hemorrhage may also be noted [13].

Histopathology and Ultrastructural Pathology

Central Nervous System: The hallmark histopathological lesion in the CNS is a non-suppurative encephalitis, characterized by neuronal degeneration and necrosis, perivascular cuffing with lymphocytes (predominantly CD3-positive T cells), gliosis, and focal hemorrhages [4, 45]. The cerebrum, cerebellum, and brainstem are all affected. Ultrastructurally, neurons are the primary target cells, with viral particles observed within the cisternae of the rough endoplasmic reticulum (RER) and Golgi apparatus [45]. Infected neurons exhibit degenerative changes, including the progressive dissolution of membranous organelles, swelling of mitochondria, and formation of autophagic vacuoles [45]. Marked swelling of astrocytic foot processes is a prominent feature, indicating disruption of the blood-brain barrier [45]. Microglial activation is evident, with phagocytosis of degenerating neurons and cellular debris [45]. Myelin lesions, including splitting and vacuolation of myelin sheaths, are also observed [45]. These ultrastructural findings provide a detailed cytopathological basis for the severe neurological dysfunction observed clinically.

Reproductive System: In the ovary, histopathology reveals extensive follicular degeneration, with hemorrhage, necrosis of the follicular epithelium, and infiltration of inflammatory cells [13, 39]. Thecal and granulosa cell layers are disrupted. In the testes, DTMUV infection induces apoptosis of spermatogenic cells, mediated by the cytochrome c (Cytc)-mitochondrial apoptotic signaling pathway [44]. Vacuolation of spermatogenic cells and testicular atrophy are observed [44].

Other Tissues: In the spleen, lymphoid depletion and necrosis are common, reflecting the immunosuppressive effects of the virus [13, 57]. The liver may show hepatocellular degeneration, necrosis, and inflammatory cell infiltration [14, 59]. The lungs can exhibit congestion, edema, and hemorrhagic lesions [14]. The kidneys may show tubular degeneration and necrosis [14].

Pathogenesis and Viral Dissemination

Following peripheral inoculation, TMUV initially replicates in local tissues, likely including muscle and skin, before disseminating via the bloodstream to target organs [25]. Viremia is detected as early as 1-2 days post-infection and peaks around 3-5 days, correlating with the onset of clinical signs [25, 57]. The virus exhibits a broad tissue tropism, with high viral loads detected in the brain, spleen, ovary, liver, kidney, and lung [13, 14, 25]. The ability of TMUV to cross the blood-brain barrier and establish infection in the CNS is a key determinant of neurovirulence. The virus likely enters the CNS via a hematogenous route, possibly through infection of brain microvascular endothelial cells, which show increased pinocytotic activity and cytoplasmic lesions during infection [45]. Once in the CNS, the virus replicates efficiently in neurons, triggering a robust inflammatory response that contributes to neuropathology [8, 45].

The molecular basis of neurovirulence is being elucidated. A single amino acid substitution in the E protein (V487A) has been identified as a critical determinant of high neurovirulence in mice, enhancing viral assembly and replication efficiency [9]. This residue is located in the transmembrane domain of the E protein, a region implicated in flavivirus particle formation [9]. Furthermore, the stem-loop I (SLI) structure in the 3' untranslated region (UTR) has been shown to regulate viral host-specific adaptation and pathogenicity in mice [40]. These findings highlight the multifactorial nature of TMUV neuropathogenesis.

Cluster-Specific Pathogenicity

The genetic diversity of TMUV is directly reflected in its pathogenic potential. Cluster 2.1 strains are generally more pathogenic in ducks than cluster 1 strains, causing higher morbidity, mortality, and more severe histopathological lesions [25]. Cluster 3 strains, particularly those of goose origin, exhibit heightened pathogenicity in geese, with mortality rates exceeding 60% in goslings [14, 19]. In contrast, cluster 3 strains may show lower pathogenicity in ducks, causing only mild and transient clinical signs [14]. This host-specific adaptation is likely driven by genetic differences in viral proteins, particularly the E protein and NS5, which influence viral entry, replication, and immune evasion [5, 9]. The emergence of cluster 3 strains with enhanced virulence in geese and the ability to infect chickens and mice underscores the dynamic and expanding host range of TMUV and the need for continued surveillance [11, 19, 22].

Diagnostic Strategies: Development and Application of Multiplex RT-qPCR

The rapid emergence and transcontinental dissemination of Tembusu virus (TMUV) since the 2010 outbreak in Chinese duck flocks have necessitated the development of robust, high-throughput molecular diagnostic platforms capable of simultaneously detecting multiple viral pathogens [13, 32, 34]. The clinical presentation of TMUV infection, characterized by acute egg-drop syndrome, neurological dysfunction, and visceral hemorrhagic lesions, overlaps considerably with other economically significant avian viral diseases, including novel duck reovirus (NDRV), duck hepatitis A virus type 1 and 3 (DHAV-1, DHAV-3), duck circovirus (DuCV), and goose astrovirus (GoAstV) [1, 51, 60]. This syndromic overlap, coupled with the high prevalence of co-infections in commercial duck and goose operations, has driven the urgent need for multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays. These assays must not only achieve exquisite sensitivity and specificity but also provide the capacity for differential diagnosis in a single reaction, thereby conserving time, reagents, and precious clinical material. The World Organisation for Animal Health (WOAH) recognizes such molecular tools as cornerstone technologies for the surveillance and control of emerging transboundary animal diseases, particularly those with the zoonotic potential increasingly attributed to TMUV [4, 38].

The Diagnostic Imperative for a Rapidly Evolving Pathogen

The biological complexity of TMUV, its division into at least three major genetic clusters (Clusters 1, 2, and 3) and multiple subclusters (2.1, 2.2, 3.1, 3.2), presents a formidable challenge for diagnostic assay design [5, 10, 17, 25]. Cluster 2.1 strains currently predominate in Thailand and Vietnam, while Cluster 3 strains have been increasingly isolated from chickens and geese in China, Taiwan, and most recently from mosquitoes and fatal dolphin cases in Thailand [4, 10, 37, 53]. Critically, these clusters exhibit significant antigenic variation and differential pathogenicity in both avian and mammalian hosts [5, 8, 9, 25]. For instance, Cluster 2.1 strains display enhanced virulence in ducks compared to Cluster 1, whereas certain Cluster 3.2 strains (e.g., GDE19-2024, HQ-22) demonstrate markedly high pathogenicity in goslings and mice, including neuroinvasive potential via intranasal inoculation [14, 19, 25, 33]. Furthermore, the expanding host range of TMUV, now documented in chickens, geese, pigeons, and importantly, bottlenose dolphins, underscores the necessity for diagnostic methods that are not only cluster-inclusive but also capable of detecting emerging variants during inter-species spillover events [4, 11, 21, 23, 39]. A monoplex assay targeting a single, highly conserved genomic region may fail to capture divergent strains or may miss co-infections that are increasingly recognized as drivers of severe pathology in commercial flocks [1, 51, 60].

Mechanistic Rationale and Assay Architecture

The foundation of an effective multiplex RT-qPCR for TMUV lies in the strategic selection of genetic targets and the meticulous optimization of primer-probe sets to ensure equable amplification efficiency across all targets. The envelope (E) protein gene and the non-structural protein 5 (NS5) RNA-dependent RNA polymerase gene have emerged as the most commonly targeted regions, owing to their sequence conservation within TMUV clusters and their divergence from other avian flaviviruses [1, 34, 60]. However, the extensive recombination and positive selection pressure acting on the NS3 and NS5 genes, as revealed by phylogeographic analyses, necessitate careful in silico validation against contemporary circulating strains [34, 62].

The landmark multiplex RT-qPCR developed by Qiu et al. (2025) exemplifies the state-of-the-art in this domain, employing a TaqMan probe-based approach for the simultaneous detection of DTMUV, NDRV, DHAV-1, and DHAV-3 [1]. The assay achieved impressive detection limits of 8.08 × 10¹ copies/μL for DTMUV, with standard curves exhibiting correlation coefficients greater than 0.99 and amplification efficiencies between 80-100% [1]. Critically, the assay demonstrated 100% specificity against a panel of other avian pathogens, including duck enteritis virus (DEV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), avian influenza virus (AIV), and Newcastle disease virus (NDV) [1]. This level of specificity is paramount given the frequent empirical misdiagnosis of TMUV on clinical grounds alone. The intra- and inter-assay coefficients of variation were maintained below 10% across a dynamic range of standard plasmid concentrations (10³ to 10⁷ copies/μL), confirming the assay's robustness for quantitative applications [1].

Similarly, Li et al. (2023) developed a duplex TaqMan qPCR targeting the conserved regions of DTMUV and GoAstV-2, achieving a lower limit of detection of 100 copies/μL for DTMUV [60]. While slightly less sensitive than the quadruplex assay, this duplex format addressed a critical diagnostic gap in goose flocks, where mixed infections with these two pathogens are particularly devastating [21, 60]. The use of different fluorophores (e.g., FAM and VIC) for each probe allows for unambiguous differential diagnosis in a single well, a feature essential for rapid clinical decision-making [60].

Comparative Sensitivity and the Emergence of Digital PCR

While conventional and multiplex RT-qPCR remain the gold standard for routine diagnostics, the advent of digital PCR (dPCR) has introduced a paradigm shift in absolute quantification. Yin et al. (2023) developed a multiplex dPCR for DTMUV, DuCV, and NDRV that demonstrated a lower detection limit of 1.3 copies/μL, a full ten-fold improvement over the equivalent multiplex qPCR [51]. The multiplex dPCR assay exhibited superior linearity and reproducibility, with intra- and inter-assay coefficients of variation as low as 0.06–1.94% [51]. When applied to 173 clinical samples, the dPCR assay yielded positivity rates for DTMUV approximately 4% higher than those obtained by qPCR, with kappa values exceeding 0.85, indicating excellent agreement while simultaneously revealing occult infections missed by less sensitive methods [51].

This heightened sensitivity is not merely an academic exercise; it has direct implications for epidemiological surveillance and the detection of persistent, low-level viral shedding. Studies have confirmed that TMUV can establish persistent infections in both avian hosts and mosquito vectors, with viral RNA detectable in tissues long after clinical resolution [16, 52]. In the context of the 2021 outbreak in North Vietnam, where 16.15% of pooled tissue samples from ducks tested positive for TMUV by conventional PCR, the application of dPCR would likely have unmasked a higher true prevalence, particularly in subclinical carriers [15]. Furthermore, the detection of TMUV RNA in fecal samples of migratory birds on Chongming Island, China, a key stopover on the East Asian-Australasian Flyway, highlights the role of sensitive molecular tools in tracking long-distance viral spread [20].

Integration with Point-of-Care and Isothermal Technologies

The diagnostic landscape for TMUV has expanded beyond traditional thermocycling platforms to include isothermal amplification technologies that facilitate field-based detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP), when coupled with CRISPR/Cas12a or Cas13a nuclease systems, provides a rapid, visual readout that is highly specific and exceptionally sensitive. Chen et al. (2024) reported an RT-LAMP-CRISPR/Cas12a assay targeting the DTMUV C gene with a detection limit of 3 copies/μL, enabling visual discrimination under blue or UV light without the need for expensive thermocyclers [47]. This approach has been further refined by integrating recombinase polymerase amplification (RPA) with CRISPR-Cas13a, achieving detection of 10² copies/μL within 50 minutes at a constant 37°C [34, 56]. The specificity of these CRISPR-based assays is remarkable; they show no cross-reactivity with other common avian viruses, and clinical validation against SYBR Green qPCR yielded 100% positive and negative predictive agreement [54].

The development of reverse transcription recombinase-aided amplification combined with lateral flow dipsticks (RT-RAA-LFD) represents another significant advance for point-of-care diagnostics. Wang et al. (2023) established an RT-RAA-LFD method for DTMUV with a detection limit of 10 copies/μL, which is 1,000-fold more sensitive than conventional RT-PCR and functionally equivalent to real-time qPCR [61]. When tested on 58 clinical samples, the RT-RAA-LFD method detected 5 positive samples compared to 4 by qPCR, underscoring its potential for identifying low-titer infections in field settings [61]. These isothermal platforms are particularly valuable in resource-limited regions where TMUV is endemic, such as Southeast Asia, where access to sophisticated laboratory infrastructure is often constrained [15, 31, 37].

Biological Significance and Diagnostic Integration

The successful deployment of multiplex RT-qPCR assays has yielded critical insights into TMUV epidemiology that would have been unattainable with single-target methods. In a survey of 215 clinical samples, Qiu et al. (2025) reported DTMUV positivity rates of 19.30%, but more importantly, they documented co-infection rates of 5.58% for DHAV-3 and DTMUV, and 0.05% for triple infections involving DTMUV, DHAV-3, and NDRV [1]. These findings corroborate the growing recognition that clinical disease in duck flocks is rarely attributable to a single etiological agent. The ability to quantify each pathogen's load within a co-infected host is essential for understanding viral interference, synergism, and the role of immunomodulation in disease progression. For example, TMUV non-structural proteins NS2B and NS4A induce autophagy and antagonize type I interferon signaling, potentially creating a permissive environment for secondary viral invaders [24, 35, 43]. Conversely, the host restriction factor TRIM14 targets the TMUV NS1 protein for proteasomal degradation while simultaneously enhancing TBK1-mediated IFN-β production, illustrating a complex host-pathogen tug-of-war that only multiplex diagnostics can fully elucidate [2].

The diagnostic community must also remain vigilant to the rapid genetic drift of TMUV. The recent identification of a novel Cluster 3.2 strain from geese in Anhui Province (AHWH) with unique E protein mutations at residues 149 and 157, and the repeated isolation of mosquito-derived strains bearing mutations in the E, NS1, NS4A, NS4B, and NS5 genes, underscore the need for periodic re-evaluation of primer and probe binding sites [10, 14, 41, 52]. Reverse genetics studies have demonstrated that single amino acid substitutions in the E protein transmembrane domain (e.g., V487A) can dramatically enhance viral replication and neurovirulence in mammalian models, a phenotypic change that may also alter the affinity of diagnostic probes [9]. The integration of multiplex RT-qPCR with next-generation sequencing surveillance programs, such as those established by Fang et al. (2023) on Chongming Island, provides a dual-pronged strategy for detecting both known and emergent TMUV strains while simultaneously quantifying viral load in complex sample matrices [20].

In conclusion, the development and application of multiplex RT-qPCR assays for TMUV represent a triumph of veterinary molecular diagnostics. From high-throughput quadruplex TaqMan platforms capable of discriminating between four major duck pathogens to ultrasensitive digital PCR and field-deployable CRISPR-based systems, the diagnostic armamentarium for TMUV is now both versatile and robust. These tools are indispensable not only for the timely diagnosis and management of clinical outbreaks but also for foundational research into viral pathogenesis, host immune responses, and the ecology of this emerging flavivirus in an era of unprecedented global change.

Host Restriction Factors and Interferon-Mediated Antiviral Responses

The intricate battle between Tembusu virus (TMUV) and its avian hosts represents a paradigm of host-pathogen co-evolution, wherein the host deploys an arsenal of restriction factors and interferon (IFN)-mediated signaling cascades to curtail viral replication, while the virus counter-evolves sophisticated evasion strategies. The type I interferon system, constituting the first line of defense against flaviviral invasion, is exquisitely orchestrated by pattern recognition receptors (PRRs) that detect viral molecular patterns and subsequently trigger a transcriptional program aimed at establishing an antiviral state [13, 74]. In the context of TMUV infection, the host's capacity to mount a robust IFN response is a critical determinant of disease outcome, with age-related variations in immune competence directly correlating with disease severity [48, 57]. This section provides an exhaustive examination of the host restriction factors that directly antagonize TMUV replication, the signaling pathways governing IFN induction, the downstream interferon-stimulated gene (ISG) effectors, and the viral countermeasures that subvert these host defenses.

Tripartite Motif (TRIM) Proteins as Potent Restriction Factors Against TMUV

The tripartite motif (TRIM) family of proteins has emerged as a cornerstone of intrinsic antiviral immunity, functioning through diverse mechanisms including ubiquitination, autophagy, and direct sequestration of viral components. Among these, duck TRIM14 (duTRIM14) has been identified as a particularly potent host restriction factor against TMUV, operating through a previously uncharacterized dual-action mechanism that simultaneously targets viral proteins for degradation and amplifies the host interferon response [2]. Specifically, duTRIM14 directly interacts with the TMUV nonstructural protein 1 (NS1), facilitating its K27/K29-linked polyubiquitination and subsequent proteasomal degradation. The critical residue Lys141 on NS1 was identified as indispensable for this process; mutagenesis of this residue significantly enhanced viral replication both in vitro and in vivo, underscoring the functional importance of this restriction axis [2]. Concurrently, duTRIM14 engages duck TBK1 (duTBK1), promoting its K63-linked polyubiquitination at residues Lys30 and Lys401, which substantially augments IFN-β production during TMUV infection [2]. This dual-action mechanism, degrading a key viral immune evasion protein while simultaneously potentiating the very signaling pathway NS1 typically suppresses, represents a remarkably sophisticated host defense strategy. The identification of duTRIM14's role provides critical mechanistic insight, as NS1 is known to be a multifunctional protein that modulates host immune responses and is itself a target for monoclonal antibody development [65].

Complementing the actions of TRIM14, the E3 ligase membrane-associated RING finger 6 (MARCH6) has been uncovered as another crucial restriction factor that targets the TMUV RNA-dependent RNA polymerase, NS5, for degradation [3]. Remarkably, MARCH6 operates through an E3 ligase activity-independent mechanism, revealing a previously uncharacterized host defense paradigm. MARCH6 expression is significantly upregulated during TMUV infection across multiple duck cell lines, suggesting a conserved and inducible cellular response. Mechanistically, MARCH6 recruits the autophagic cargo receptor TOLLIP, which facilitates the NS5-TOLLIP interaction independent of conventional ubiquitin signaling, subsequently directing NS5 to phagophores for selective autophagic degradation [3]. This MARCH6-NS5-TOLLIP axis highlights the importance of selective autophagy as a fundamental antiviral mechanism. The targeting of NS5 is particularly strategic, as NS5 is the most conserved flaviviral protein, serving as both the RNA-dependent RNA polymerase and a methyltransferase essential for capping viral RNA. The methyltransferase activity of NS5, particularly residue E218, is critical for viral replication and cap methylation, and its mutation impairs translation while triggering RIG-I-like receptor signaling [62]. By degrading NS5, MARCH6 effectively halts viral genome replication at its core.

The Interferon Signaling Cascade: From Pattern Recognition to ISG Induction

The induction of type I interferons (IFN-α/β) and type III interferons (IFN-λ) is the central event in the antiviral innate immune response to TMUV. Duck type I and type III IFNs both exhibit potent antiviral activity against the virus, though with distinct kinetics. Recombinant duck IFN-β (duIFN-β) demonstrates faster and more potent induction of interferon-stimulated genes (ISGs) such as ZAP, OAS, and RNaseL in vitro and in vivo compared to duck IFN-λ (duIFN-λ), correlating with superior antiviral efficacy against DTMUV in ducks [64]. Both interferons activate the ISRE (interferon-stimulated response element) promoter and establish an antiviral state, but the kinetic advantage of type I IFN may be crucial for controlling the rapid replication cycle of TMUV [64].

The signaling cascade initiating IFN production is triggered by host PRRs that recognize TMUV RNA. The RIG-I-like receptors (RLRs), RIG-I, MDA5, and LGP2, play pivotal roles in sensing flaviviral RNA. Duck LGP2 (duLGP2) has been characterized as a negative regulator of the type I IFN response during the resting state and early stages of TMUV infection, exhibiting a context-dependent role [68]. Overexpression of duLGP2 significantly reduces the cell's antiviral capacity by inhibiting type I IFN production and downstream ISG expression, while its knockdown exerts the opposite effect [68]. This regulatory complexity ensures that the interferon response is tightly controlled to prevent immunopathology while still mounting an effective antiviral state. Downstream of RLR activation, the mitochondrial antiviral-signaling protein (MAVS) serves as a central adaptor, integrating signals from RIG-I and MDA5. The critical nature of MAVS in the anti-TMUV response is underscored by the fact that the virus has evolved mechanisms to specifically target this molecule for degradation, as discussed later.

Interferon regulatory factors (IRFs) are the master transcriptional regulators of IFN expression. Avian IRF1 and IRF7 play overlapping yet distinct roles in regulating both IFN-dependent and IFN-independent antiviral responses to TMUV [71]. While both IRF1 and IRF7 inhibit TMUV replication primarily through the regulation of type I IFN expression, IRF1 can directly promote the expression of specific ISGs such as VIPERIN and CMPK2 in an IFN-independent manner when IRF7 and type I IFN signaling are compromised [71]. This functional redundancy provides a failsafe mechanism ensuring that antiviral responses can be mounted even if one arm of the signaling pathway is inhibited. The IRF7-mediated activation of IFN-β is itself subject to tight regulation via ubiquitination; the E3 ubiquitin ligase RNF123 targets the suppressor of cytokine signaling 1 (SOCS1) for K48-linked ubiquitination and proteasomal degradation, thereby relieving SOCS1-mediated inhibition of IRF7 and promoting TLR3- and IRF7-induced type I IFN production during DTMUV infection [69].

Viral Countermeasures: Antagonism of the Interferon System by TMUV Nonstructural Proteins

Given the potency of the interferon response, TMUV has evolved an array of sophisticated mechanisms to antagonize host innate immunity. The viral nonstructural protein 2B (NS2B) has been identified as a key negative regulator of IFN induction. TMUV NS2B specifically interacts with duck MAVS (duMAVS), recruiting the E3 ubiquitin ligase duck membrane-associated RING-CH-type finger 5 (duMARCH5) to modify duMAVS via K48-linked polyubiquitination, leading to its proteasomal degradation [35]. This effectively dismantles the RLR signaling platform at its most critical junction, preventing downstream activation of IRF3/IRF7 and subsequent IFN-β production. The residues K321, K354, K398, and K411 on duMAVS were identified as crucial for NS2B-mediated ubiquitination and degradation [35]. This mechanism is directly confirmed by findings that NS2B can inhibit the induction of IFN-β mRNA triggered by either Newcastle disease virus infection or poly(I:C) treatment [71].

Beyond NS2B, the NS5 protein of TMUV employs a distinct strategy to subvert the IFN system by hijacking TRAF3 (TNF receptor-associated factor 3), a key signaling molecule in the RIG-I pathway. DTMUV infection enhances duTRAF3 expression, and overexpression of duTRAF3 inhibits DTMUV replication in a dose-dependent manner by activating the transcriptional activity of IFN-α and its downstream ISGs [26]. However, TMUV NS5 directly interacts with duTRAF3 and inhibits its expression, thereby neutralizing this antiviral signaling node [26]. This dual targeting of MAVS and TRAF3 ensures that the RLR signaling cascade is disrupted at multiple points.

A more elaborate negative-feedback regulatory pathway involving JOSD1, SOCS1, and IRF7 further demonstrates the complexity of TMUV's immune evasion strategy. DTMUV infection activates Toll-like receptor 3 (TLR3) signaling, which upregulates SOCS1 [36]. The deubiquitinase JOSD1 then stabilizes SOCS1 by binding to its SH2 domain and mediating its deubiquitination, preventing SOCS1's own proteasomal degradation [36]. The stabilized SOCS1 then acts as an E3 ubiquitin ligase, binding to IRF7 through its SH2 domain and mediating K48-linked ubiquitination and proteasomal degradation of IRF7, ultimately inhibiting IRF7-mediated type I IFN production and promoting viral proliferation [36]. This JOSD1-SOCS1-IRF7 axis represents a sophisticated viral strategy to exploit host regulatory machinery to suppress the central transcriptional activator of the IFN response.

The N-myc and STAT interactor (NMI) adds another layer to this intricate regulatory network. While NMI is an interferon-induced protein, duck NMI (duNMI) has been shown to inhibit the antiviral response during DTMUV infection. Overexpression of duNMI inhibits DTMUV replication, but paradoxically, it does so by inhibiting the transcriptional activity of IFN-I related cytokines [18]. Specifically, duNMI interacts with duck IRF7 through its NID1 and NID2 domains, inhibiting IRF7 expression and the subsequent activation of IFN-β [18]. This suggests that NMI may act as a feedback inhibitor, preventing excessive or prolonged IFN signaling that could be detrimental to the host.

ISG Effectors and Their Role in Restricting TMUV Replication

The interferon-induced antiviral state is ultimately executed by hundreds of ISGs that target various stages of the viral life cycle. Transcriptomic analyses of DTMUV-infected duck embryo fibroblasts (DEFs) have revealed dynamic changes in gene expression, with 3129 differentially expressed genes identified across the infection time course, many of which are associated with host immunity and virus infection [74]. The ISGs VIPERIN, IFIT5, CMPK2, and MX1 have been specifically implicated in restricting TMUV replication, with their expression being regulated by both IRF1 and IRF7 [71]. The zinc-finger antiviral protein (ZAP), 2'-5'-oligoadenylate synthetase (OAS), and ribonuclease L (RNaseL) are also induced by duck type I and type III IFNs and contribute to the degradation of viral RNA [64].

The involvement of cellular proteins in the restriction of TMUV extends beyond canonical ISGs. Vimentin, an intermediate filament protein, has been shown to act as a negative regulator of DTMUV replication. DTMUV infection induces vimentin rearrangement, which is dependent on CDK5-mediated phosphorylation at Ser56 [63]. This rearrangement negatively modulates viral replication, suggesting that the host cytoskeleton can be repurposed as an antiviral scaffold [63]. Conversely, the heat shock protein 70 (HSP70) is co-opted by TMUV to promote its replication; TMUV infection induces HSP70 expression, and both inhibition and antibody-mediated neutralization of HSP70 significantly reduce viral infectivity and progeny production [72]. The ribosomal protein S14 (RPS14) also functions as a restriction factor, as its overexpression decreases DTMUV replication [66]. However, the virus counters this by upregulating host miR-146b-5p, which targets and downregulates RPS14, thereby promoting viral replication [66].

Cellular autophagy represents a double-edged sword in TMUV infection. The virus induces both complete and incomplete autophagy depending on the cell type. TMUV nonstructural proteins NS2B and NS4A induce complete autophagy to facilitate viral replication by interacting with the polyubiquitin-binding protein SQSTM1/p62, while NS3 induces autophagy through the ERK2 and PI3K/AKT-mTOR signaling pathways [24, 70]. In neuronal cells, DTMUV induces incomplete autophagy via the ERK/mTOR and AMPK/mTOR signaling pathways to promote viral replication, contributing to neuropathogenesis [43]. The pharmacological induction of autophagy with rapamycin can enhance TMUV replication by up to 15-fold, highlighting the critical role of this degradative pathway in the viral life cycle [24].

Differential Antiviral Responses Across Host Species and Tissues

The outcome of TMUV infection is profoundly influenced by host age, species, and tissue-specific innate immune responses. Adult laying ducks (27-week-old) mount a robust neutralizing antibody response and exhibit a significant negative correlation between cytotoxic T lymphocyte (CTL) responses and reduction of viremia, in contrast to younger ducks (4-week-old) where myeloid cell responses are implicated in disease progression [48, 57]. The identification of Anpl-UAA*76-restricted CTL epitopes of TMUV in inbred HBW/B4 ducks has provided a molecular basis for understanding duck anti-TMUV CTL immunity, with conserved TMUV peptides capable of stimulating IFN-γ secretion and lymphocyte proliferation [73].

In the mosquito vector, the innate immune response also shapes viral transmission dynamics. Cluster 1 and Cluster 2.1 DTMUV strains induce differential expression of macroglobulin complement-related factor (MCR), thioester-containing protein, and antimicrobial peptides in the salivary glands of Culex tritaeniorhynchus [5]. Furthermore, a 34-kDa salivary protein in Aedes albopictus enhances DTMUV infectivity in salivary glands by suppressing the antiviral immune response, particularly through modulation of MCR and antimicrobial peptide production [46]. This vector-virus interplay underscores the importance of innate immunity at the arthropod-host interface.

The expanding host range of TMUV, including documented natural fatal infections in bottlenose dolphins and serological evidence in duck farm workers, highlights the zoonotic potential of this virus and the need for continued surveillance under a One Health framework [4, 38]. Cluster 3 TMUV strains have demonstrated the ability to cause severe neurological disease and mortality in mice via intranasal infection, raising concerns about airborne transmission to mammals [19]. The mouse model has proven invaluable for differentiating neurovirulence between TMUV clusters, with enhanced neurovirulence of cluster 2.1 relative to cluster 2.2 being associated with more efficient replication in the central nervous system and elevated expression of IFN-β, IL-1β, IL-6, TNF-α, Ifit1, and Ifit2 [8]. This positive correlation between TMUV neurovirulence and the magnitude of the antiviral innate immune response suggests that excessive inflammation may contribute to pathology, a finding consistent with the M1 polarization of chicken macrophages via the MyD88-NF-κB signaling pathway [67]. The intricate balance between protective immunity and immunopathology remains a central theme in TMUV pathogenesis.

Co-infections and Mixed Viral Pathologies in Duck Populations

The clinical and pathological landscape of Tembusu virus (TMUV) infection in domestic waterfowl is profoundly complicated by the frequent occurrence of co-infections with other avian pathogens. This reality reflects the intensive farming conditions prevalent across Asia, where high-density duck populations are concurrently exposed to multiple viral, bacterial, and parasitic agents. The economic toll exacted by TMUV is often amplified by these mixed infections, which can obscure diagnosis, exacerbate clinical outcomes, and complicate control strategies. A comprehensive understanding of the co-infection ecology of TMUV is thus essential for accurate disease surveillance, effective vaccine deployment, and rational therapeutic intervention. This section synthesizes the available evidence on the incidence, detection, and biological implications of co-infections involving TMUV, with a particular focus on duck hepatitis A viruses, novel duck reovirus, duck circovirus, goose astrovirus, and other pathogens that frequently co-circulate in affected flocks.

The Ubiquity of Mixed Viral Infections in Commercial Duck Flocks

The commercial duck industry, particularly in China and Southeast Asia, operates under biosecurity conditions that often permit the simultaneous circulation of multiple viral pathogens. The emergence of TMUV in 2010 [13, 34] added a new dimension to an already complex infectious disease ecology. Epidemiological surveys utilizing advanced multiplex molecular tools have consistently revealed that TMUV is frequently not the sole pathogen present in clinical specimens. This phenomenon is not merely a laboratory curiosity; it has tangible consequences for disease severity, tissue tropism, and the immunological trajectory of infection. For instance, a large-scale study employing a TaqMan probe-based multiplex quantitative real-time RT-PCR (RT-qPCR) for the simultaneous detection of duck Tembusu virus, novel duck reovirus (NDRV), duck hepatitis A virus type 1 (DHAV-1), and duck hepatitis A virus type 3 (DHAV-3) in 215 clinical samples revealed striking co-infection patterns [1]. The positivity rate for TMUV alone was 19.30%, but co-infection with DHAV-3 was observed in 5.58% of samples, and a triple co-infection involving DHAV-3, TMUV, and NDRV was detected in 0.05% of cases [1]. These data underscore that mixed infections are not rare events but rather constitute a significant proportion of field cases.

The diagnostic challenge posed by these co-infections cannot be overstated. Clinical signs such as egg-drop syndrome, neurological dysfunction, and visceral hemorrhage can be induced by TMUV, DHAV, NDRV, and other agents, making syndromic diagnosis unreliable. The development of multiplex detection platforms has therefore been a critical priority. Beyond the quadruplex RT-qPCR [1], other sophisticated assays have been validated for co-detection. A multiplex digital PCR (dPCR) targeting TMUV, duck circovirus (DuCV), and NDRV demonstrated superior sensitivity compared to conventional qPCR, with detection limits as low as 1.3 copies/μL and positive detection rates of 18.5% for TMUV, 29.5% for DuCV, and 14.5% for NDRV in 173 clinical samples [51]. The kappa values of 0.85–0.89 between dPCR and qPCR indicated excellent agreement, but the higher sensitivity of dPCR revealed a greater burden of co-infections than previously appreciated [51]. Similarly, a duplex TaqMan qPCR for TMUV and goose astrovirus genotype 2 (GoAstV-2) was developed specifically to address the problem of mixed infections in goose flocks, highlighting the clinical importance of this particular co-pathology [60]. The lower limits of detection for this assay were 100 copies/μL for TMUV and 10 copies/μL for GoAstV-2, with high specificity against other common avian viruses [60]. These diagnostic advancements are not merely technical achievements; they are foundational for understanding the true epidemiological footprint of TMUV in mixed-infection contexts.

Documented Co-infections with Hepatotropic and Enteric Viruses

Among the most frequently documented co-infections involving TMUV are those with duck hepatitis A viruses (DHAV-1 and DHAV-3) and goose astrovirus. DHAV is a picornavirus that causes acute hepatitis in ducklings, with mortality rates that can approach 100% in young birds. The co-circulation of DHAV with TMUV creates a particularly dangerous scenario, as both viruses target visceral organs and can induce severe hepatic and neurological damage. The multiplex RT-qPCR study mentioned above provides the most explicit data on this co-infection, reporting that 5.58% of DTMUV-positive samples also harbored DHAV-3, a genotype that has emerged as a significant pathogen in Chinese duck flocks [1]. While the study did not perform detailed pathological comparisons, the concurrent presence of these two viruses likely potentiates hepatic injury and systemic inflammation. DTMUV is known to induce apoptosis and endoplasmic reticulum stress via NS3- and NS5-mediated pathways [28, 74], while DHAV directly lyses hepatocytes. The combined cytopathic effect could overwhelm the regenerative capacity of the liver, leading to fulminant hepatitis and increased mortality. This is particularly relevant for ducklings, in which DHAV infection is already a major cause of death. The triple infection involving DHAV-3, DTMUV, and NDRV, though observed at a lower frequency (0.05%) [1], represents an even more severe scenario, as NDRV is also known to cause hemorrhagic and necrotic lesions in multiple organs.

The co-infection of TMUV with goose astrovirus (GoAstV) is of particular concern in goose populations. GoAstV genotype 2 is a causative agent of gout and visceral urate deposition in goslings, a disease that has caused substantial losses in China. The development of a duplex qPCR for TMUV and GoAstV-2 was explicitly motivated by the recognition that mixed infections of these two viruses are an important problem in the goose industry [60]. The pathogenesis of this co-infection may involve synergistic disruption of renal function and purine metabolism, though detailed mechanistic studies are lacking. The detection of TMUV in geese has become increasingly common, with cluster 3 strains being isolated from geese with ovaritis, pancreatic necrosis, and neurological signs [10, 14, 19, 21, 27, 41, 49, 59]. The GDZQ2022 strain, a subgenotype 3 TMUV isolated from geese in Guangdong, exhibited high pathogenicity in goose embryos but relatively mild virulence in goslings, with low mortality despite severe histopathological lesions in the brain, liver, and spleen [59]. When co-infection with GoAstV occurs, the clinical picture may shift dramatically, as the astrovirus-induced metabolic derangements could exacerbate the TMUV-induced tissue damage. The lack of systematic co-infection studies in geese represents a critical gap in the literature, particularly given the expanding host range of cluster 3 TMUV strains [11, 14, 22, 39].

Reoviruses and Circoviruses: Immunosuppressive Synergies and Diagnostic Overlap

The co-occurrence of TMUV with novel duck reovirus (NDRV) and duck circovirus (DuCV) introduces a dimension of immunological complexity that is only beginning to be understood. NDRV, a member of the Reoviridae family, causes hemorrhagic necrotic hepatitis and splenic necrosis in ducks, with clinical signs that can mimic those of severe TMUV infection. The multiplex dPCR study by Yin et al. [51] found that 14.5% of clinical samples were positive for NDRV, and co-infection with TMUV and DuCV was also documented. While the study did not report the specific rates of TMUV–NDRV dual positivity, the concurrent circulation of these viruses is well-established [1]. The pathological synergy between TMUV and NDRV may be driven by their shared tropism for lymphoid and reticuloendothelial tissues. NDRV is known to cause immunosuppression through lymphoid depletion, which could predispose ducks to more severe TMUV replication and dissemination. Conversely, TMUV-induced apoptosis of immune cells [28, 57] could create a permissive environment for NDRV expansion. This reciprocal potentiation has not been formally tested in experimental co-infection models, but the epidemiological association warrants urgent investigation.

Duck circovirus (DuCV) is a small, single-stranded DNA virus that is a classic immunosuppressive pathogen. DuCV infection is associated with feathering disorders, growth retardation, and secondary bacterial and viral infections. The high prevalence of DuCV (29.5%) reported in the multiplex dPCR survey [51] is striking, particularly given that TMUV was detected in 18.5% of the same sample set. The authors did not explicitly calculate the co-infection rate between DuCV and TMUV, but the individual prevalence figures suggest a substantial overlap. DuCV targets the bursa of Fabricius and lymphoid tissues, inducing apoptosis and lymphocytic depletion [51]. This immunosuppression could profoundly alter the outcome of TMUV infection. In ducks with subclinical DuCV, TMUV challenge might result in higher viremia, more prolonged shedding, and enhanced neuroinvasiveness. The established mechanisms by which TMUV evades innate immunity, including NS2B-mediated degradation of MAVS [35], NS5-mediated suppression of TRAF3 signaling [26], and JOSD1-SOCS1-mediated inhibition of IRF7 [36], could be further amplified in an immunosuppressed host. The convergence of TMUV's immune evasion arsenal with DuCV-induced lymphocyte depletion represents a hypothetical but plausible pathway to severe, disseminated disease. Diagnostic assays that can reliably distinguish these pathogens are therefore not just academic tools but essential components of a rational disease management strategy. The specificity of the multiplex dPCR against Muscovy duck reovirus, MDPV, GPV, H4 AIV, H6 AIV, and NDV [51] ensures that TMUV is not falsely attributed to other agents in co-infected samples.

Genotypic and Host-Driven Variations in Co-infection Dynamics

Not all TMUV strains are equally likely to participate in co-infections, nor do all host species respond identically. The genetic diversity of TMUV, which is currently classified into three major clusters (1, 2, and 3) with further subclusters [5, 13, 17, 34], has been linked to differences in pathogenicity, tissue tropism, and host range. Cluster 1 strains, for instance, are generally less pathogenic in ducks than cluster 2.1 strains, with delayed viremia, lower viral loads, and milder pathological changes [25]. One might hypothesize that less virulent strains are more likely to persist as subclinical co-infections, whereas highly virulent cluster 2.1 or 3 strains would dominate the clinical picture and mask the presence of other pathogens. However, the available data do not yet support such a clear stratification. The multiplex RT-qPCR study [1] did not perform genotyping on the TMUV strains detected, so it is unknown whether the co

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